Solution Structure of an Archaeal RNase P Binary Protein Complex: Formation of the 30-kDa Complex between Pyrococcus furiosus RPP21 and RPP29 Is Accompanied by Coupled Protein Folding and Highlights Critical Features for Protein–Protein and Protein–RNA Interactions

Abstract

Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) enzyme that catalyzes the Mg 2+-dependent 5′ maturation of precursor tRNAs. In all domains of life, it is a ribozyme: the RNase P RNA (RPR) component has been demonstrated to be responsible for catalysis. However, the number of RNase P protein subunits (RPPs) varies from 1 in bacteria to 9 or 10 in eukarya. The archaeal RPR is associated with at least 4 RPPs, which function in pairs (RPP21–RPP29 and RPP30–POP5). We used solution NMR spectroscopy to determine the three-dimensional structure of the protein–protein complex comprising Pyrococcus furiosus RPP21 and RPP29. We found that the protein–protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies surfaces rich in conserved basic residues likely vital for recognition of the RPR and/or precursor tRNA. Furthermore, enzymatic footprinting experiments allowed us to localize the RPP21–RPP29 complex to the specificity domain of the RPR. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme.